Rabies virus (RV) is a non-segmented negative-strand RNA virus within the Rhabdoviridae family and lyssavirus genera. The RV genome is about 12-kb in size and encodes five monocistronic RNAs encoding the nucleocapsid protein (N), phosphoprotein (P), matrix protein (M), the transmembrane glycoprotein (G), and the viral polymerase (L). The RV N protein encapsidates the viral RNA to form the ribonucleoprotein (RNP), which is the template for RNA transcription and replication by the viral polymerase-complex composed of the P and L proteins. The RV M bridges the RNP with the cytoplasmic domain (CD) of RV G in the host cell-derived viral membrane. The RV G mediates infection of the host cell. The main feature of rabies virus is neuroinvasiveness, which refers to its unique ability to invade the central nervous system (CNS) from peripheral sites.
Rabies virus is a promising vaccine vector able to induce humoral and cellular immune responses efficiently to foreign antigens. Recombinant live-viral vectors expressing foreign antigens efficiently induce potent cellular and humoral immune responses against the expressed antigens. Because of low seroprevalence in the human population, RV is an excellent viral vector candidate. Methods for engineering the virus are well established, up to two foreign genes totaling 6.5 kb have been incorporated thus far, and foreign sequences are stably maintained. RV grows to high titers in cell lines approved for human vaccine production and manufacture is economical. See, Smith et al., 2006, Virology, 353(2): 344-356. For example, replication-competent RV comprising heterologous nucleic acids sequences encoding the HIV-1 gp160 is described in WO 01/55330. Immunization with RV encoding bacterial, viral or cancer antigens, fused to at least a portion of the RV N protein or G protein is described in US Pat. Pub. 2008/0311147. Expression of HIV-1 Env or Gag results in potent immune responses directed against HIV-1 (Schnell et al, 2000, Proc. Natl. Acad. Sci USA 97(7): 3544-3549).
The availability of reverse genetics technology, has allowed the modification of RV viral elements that account for pathogenicity and immunogenicity, and has made the systematic development of safer and more potent modified-live rabies vector feasible. For example, the pathogenicity of fixed RV strains (i.e., ERA, SAD) can be completely abolished for immunocompetent mice by introducing single amino acid exchanges in their G protein (Faber et al., 2005, J Virol 79:14141-14148). RVs containing a SADB19 G with an Arg333→Glu333 mutation are nonpathogenic for adult mice after intracranial/intracerebral inoculation; an Asn194→Ser194 mutation in the same gene prevents the reversion to pathogenic phenotype (Faber et al., 2005, J Virol 79:14141-14148; Dietzschold et al., 2004, Vaccine 23:518-524; U.S. Pat. No. 7,695,724). The G gene containing both mutations has been designated as “GAS”. Using the GAS gene, the single and double GAS RV variants, SPBNGAS and SPBNGAS-GAS, respectively, were constructed (Faber et al., 2005, J Virol 79:14141-14148; Li et al., 2008, Vaccine 26:419-426). The introduction of a second G gene significantly improves the efficacy of the vaccine by enhancing its immunogenicity through higher expression of G (Faber et al., 2002, J Virol 76:3374-3381). Elevated G expression is associated with the strong up-regulation of genes related to the NFκB signaling pathway, including IFN-α/β and IFN-γ (Li et al., 2008, Vaccine 26:419-426) and increased cell death (Faber et al., 2002, J Virol 76:3374-3381). Furthermore, the presence of two G genes also decreases substantially the probability of reversion to pathogenicity because the nonpathogenic phenotype determined by GAS is dominant over a pathogenic G that could emerge during virus growth in vivo or in vitro (Faber et al., 2007, J Virol 81:7041-7047).
A further improvement in recombinant RV safety is the highly attenuated triple RV G variant, SPBAANGAS-GAS-GAS (Faber et al., 2009, Proc. Natl. Acad. Sci USA 206(27):11300-11305). The SPBAANGAS-GAS-GAS variant is completely nonpathogenic after intracranial infection of mice that are either developmentally immunocompromised (e.g., 5-day-old mice) or mice that have inherited deficits in immune function.
Recombinant RVs that express foreign antigens derived from various disease-causing agents may serve as useful vaccine vectors. However, a problem that often arises with the use of recombinant viruses in vaccinology is that they are designed such that the immune system is exposed to antigens of the virus vector and foreign agent coincidentally, and the immune response against the virus vector dominates over the response to the foreign antigen.
Adjuvants have been expensively used to improve the potency of vaccines. Safety and tolerability are critical regulatory issues confronting adjuvant use. The field of adjuvant development is reviewed by Petrovsky et al., “New-Age Vaccine Adjuvants: Friend or Foe?” BioPharm International, Aug. 2, 2007, http<colon>//biopharminternational<dot>findpharma<dot>com/biopharm/article/articleDetail<dot>jsp?id=444996&sk=&date=&pageID=5.
As described by Petrovsky et al., the benefits of incorporating any adjuvant into vaccines must be balanced against any increased reactogenicity or risk of adverse reactions. In most cases, increased adjuvant potency is associated with increased reactogenicity and toxicity. For example, while complete Freund's adjuvant (CFA) is the “gold standard” in terms of adjuvant potency, its extreme reactogenicity and toxicity precludes its use in human vaccines.
As described by Petrovsky et al., a major unsolved challenge in adjuvant development is how to achieve a potent adjuvant effect while avoiding reactogenicity or toxicity. Most newer human adjuvants including MF59,4 ISCOMS,5 QS21,6 AS02,7 and ASO48 have substantially higher local reactogenicity and systemic toxicity than alum. Even alum, despite being FDA-approved, has significant adverse effects including injection site pain, inflammation, and lymphadenopathy, and less commonly injection-site necrosis, granulomas, or sterile abscess. Although many adjuvant-caused vaccine reactions are not life-threatening and do resolve over time, they remain one of the most important barriers to better community acceptance of routine prophylactic vaccination. This particularly applies to pediatric vaccination where prolonged distress in the child due to increased reactogenicity may lead directly to parental and community resistance to vaccination.
Use of oil-in-water emulsions has been limited by their reactogenicity and potential for adverse reactions. Oil-in-water particles are irritants and cause local inflammation, inducing a chemotactic signal that elicits local macrophage invasion. Because of frequent adverse reactions, the major human use of oil-in-water emulsions has been in therapeutic cancer and HIV vaccines. Petrovsky et al., supra. Other adjuvants proposed for human use are characterized b varying degrees of safety concerns and/or reactogenicity risks: monophosphoryl Lipid A (significant reactogenicity); unmethylated CpG dinucleotide (overall, reactogenicity, toxicity, including site reactions, flu-like symptoms, and headache), QS21, comprising triterpenoid glycosides (saponins) derived from the bark of the South American soap bark tree (severe injection site pain, granulomas, and severe hemolysis); ISCOMs, which are immunostimulating complexes containing a saponin, a sterol, and, optionally, a phospholipid (toxicity, and safety concerns). Petrovsky et al., supra. Only recently, a nanocrystalline particles of inulin, Advax, has shown promising freedom from side effects, the adjuvant is a natural plant-derived polysaccharide consisting of a chain of fructose molecules ending in a single glucose.
What are needed are recombinant RV-based compositions and immunization methods that provide for a vigorous immune response to foreign antigens expressed by the recombinant RVs, without the use of adjuvants.